Of the many fascinating discoveries made by genome sequencing projects, perhaps none is more provocative than the realization that eukaryotic and prokaryotic organisms universally possess a huge number of uncharacterized enzymes. This finding belies the commonly held notion that our knowledge of cell metabolism is near completion and underscores the vast landscape of unannotated metabolic and signaling networks that operate in our cells and tissues. The functional annotation of uncharacterized enzymatic pathways, thus, represents a grand challenge for researchers in the post-genomic era. To achieve this goal, selective pharmacological tools to perturb these pathways would be of particular value. A pressing question, however, immediately arises: how can one rapidly and systematically develop potent and selective small-molecule inhibitors for uncharacterized enzymes? Here, we propose to solve this key problem using an innovative chemical proteomic technology termed activity-based protein profiling (ABPP). Specifically, we will develop a streamlined, "library-versus-library" platform for competitive ABPP that enables the parallel discovery of inhibitors for numerous uncharacterized enzymes directly in native proteomes. As an initial application, we will use this platform to develop potent and selective inhibitors for the serine hydrolase superfamily of enzymes. The serine hydrolase class represents an excellent target for inhibitor discovery by ABPP for multiple reasons: 1) it is an exceptionally large family of enzymes (~110 members in humans) that contains numerous uncharacterized members (~40-50% of the family), 2) it is enriched in enzymes that play key roles in mammalian signaling networks, especially in the nervous system, and 3) ABPP probes and inhibitor libraries for serine hydrolases are in place to enable library-versus-library screening. If successful, the studies proposed in this application will have a profound and immediate impact on both the biological and chemical research communities. The biology community will receive a bounty of new pharmacological tools in the form of serine hydrolase inhibitors that have been optimized for potency and selectivity in vitro and in vivo. The chemistry community will receive a validated methodological process for large-scale inhibitor discovery in the form of a fully developed library-versus-library competitive ABPP platform that can be applied, in principle, to any enzyme class. We believe that this application is well-suited for the EUREKA program because: 1) it tackles n difficult technical problem of extraordinary biological and biomedical significance using cutting-edge methodologies, and 2) the magnitude of impact of our proposed studies, which should provide transforming research tools and methodologies for the biological and chemistry communities, is exceptionally high. A large fraction of drugs used to treat human disease inhibit enzymes. A huge number of uncharacterized enzymes are encoded by the human genome, suggesting that many as- of-yet undiscovered drug targets may exist in our cells and tissues. The goal of this application is to develop an innovative platform to systematically discover inhibitors for uncharacterized enzymes of relevance to human health and disease.